Publications

3843

In China, citrus Huanglongbing (HLB) disease is caused by the Candidatus Liberibacter asiaticus bacterium, which is carried by the Asian citrus psyllid Diaphorina citri Kuwayama. It was hypothesized that the epidemic of the HLB may related with the rate of bacterium presence in the insect vector and bacterium content in plant tissues, as well as the phyllosphere microbe communities changes. This study systematically analyzed the presence or absence of Ca. L. asiaticus in citrus tree leaves and in the insect vector D. citri over a 6-year period using real-time PCR. In addition, changes in the number of bacteria carried by D. citri over 12 months were quantified, as well as the relationship between the proportion of D. citri carrying Ca. L. asiaticus and the proportion of plants infected with Ca. L. asiaticus were analyzed. Results showed that the proportion of D. citri carrying bacteria was stable and relatively low from January to September. The bacteria in citrus leaves relatively low in spring and summer, then peaked in December. The proportion of D. citri carrying bacteria gradually declined from 2014 to 2019. The proportion of D. citri carrying Ca. L. asiaticus showed a significant positive correlation with the proportion of diseased citrus. The phyllosphere bacterial and fungal communities on the healthy citrus leaf were significantly different with the disease leaf in April and December. Pathogenic invasions change the citrus phyllosphere microbial community structure. It could be summarized that citrus Huanglongbing correlated with incidence of Diaphorina citri carrying Candidatus Liberibacter asiaticus and citrus phyllosphere microbiome.

Rickettsiales (Rickettsia spp., Ehrlichia spp., and Anaplasma spp., etc.) are generally recognized as potentially emerging tick-borne pathogens. However, some bacteria and areas in China remain uninvestigated. In this study, we collected 113 ticks from mammals in Guizhou Province, Southwest China, and screened for the Rickettsiales bacteria. Subsequently, two spotted fever group Rickettsia species and one Candidatus Lariskella sp. were detected and characterized. “Candidatus Rickettsia jingxinensis” was detected in Rhipicephalus microplus (1/1), Haemaphysalis flava (1/3, 33.33%), Haemaphysalis kitaokai (1/3), and Ixodes sinensis (4/101, 3.96%), whereas Rickettsia monacensis was positive in H. flava (1/3), H. kitaokai (2/3), and I. sinensis ticks (74/101, 73.27%). At least two variants/sub-genotypes were identified in the R. monacensis isolates, and the strikingly high prevalence of R. monacensis may suggest a risk of human infection. Unexpectedly, a Candidatus Lariskella sp. belonging to the family Candidatus Midichloriaceae was detected from Ixodes ovatus (1/4) and I. sinensis (10/101, 9.90%). The gltA and groEL gene sequences were successfully obtained, and they show the highest (74.63–74.89% and 73.31%) similarities to “Candidatus Midichloria mitochondrii”, respectively. Herein, we name the species “Candidatus Lariskella guizhouensis”. These may be the first recovered gltA and groEL sequences of the genus Candidatus Lariskella.

Huanglongbing (HLB) is an important citrus disease associated with the phloem-limited, uncultured bacterium ‘Candidatus Liberibacter asiaticus’(CLas). Effective treatments against CLas have to be validated in the field, however, methods for the field assessment of treatment effectiveness are time-consuming, in part because DNA-based assays, including quantitative PCR (qPCR), cannot differentiate between live and dead bacterial DNA. The aim of this study was to develop a method for rapid the evaluation of HLB therapies in field experiments. To this aim, a DNA extraction method from citrus leaf tissues with propidum monoazide (PMA), a dye that binds covalently to dsDNA making it unavailable for amplification in subsequent qPCR reactions, was optimized. The results indicated that the efficacy of PMA-qPCR was highly dependent on the primer set used. Primers targeting the 16S region of CLas showed a clear distinction between qPCR from PMA-treated and non-treated samples, while the RNR and LJ900 primers did not show significant differences between the DNA extraction methods. The PMA-qPCR viability analysis of CLas from citrus cuttings treated with different ampicillin (Amp) concentrations showed that all concentrations reduced CLas titers significantly starting 4 days after the initial treatment, unlike the water treatment, which did not show any change. This method was used for assessing the antibacterial activity of Amp, Streptomycin, Oxytetracycline (OTC), and a water control in field tests. The PMA-qPCR results indicated that Amp and OTC displayed significant antibacterial activity against CLas by 8 days post-injection, which was not detected in the non-PMA qPCR analysis. This method could allow the rapid validation of treatments against CLas in field experiments and facilitate the implementation of effective management strategies against HLB.

Anaerobic methane metabolism is among the hallmarks of Archaea, originating very early in their evolution. Here, we show that the ancestor of methane metabolizers was an autotrophic CO 2 -reducing hydrogenotrophic methanogen that possessed the two main complexes, methyl-CoM reductase (Mcr) and tetrahydromethanopterin-CoM methyltransferase (Mtr), the anaplerotic hydrogenases Eha and Ehb, and a set of other genes collectively called “methanogenesis markers” but could not oxidize alkanes. Overturning recent inferences, we demonstrate that methyl-dependent hydrogenotrophic methanogenesis has emerged multiple times independently, either due to a loss of Mtr while Mcr is inherited vertically or from an ancient lateral acquisition of Mcr. Even if Mcr is lost, Mtr, Eha, Ehb, and the markers can persist, resulting in mixotrophic metabolisms centered around the Wood-Ljungdahl pathway. Through their methanogenesis remnants, Thorarchaeia and two newly reconstructed order-level lineages in Archaeoglobi and Bathyarchaeia act as metabolically versatile players in carbon cycling of anoxic environments across the globe.